Musculoskeletal tissues are typically heterogeneous, where notable patterns of cell phenotype and matrix architecture are appear to optimally support spatial gradients in mechanical loading: chondrocytes, type II collagen, and proteoglycan in regions of high pressure; fibroblasts and type I collagen in regions of high distortion. Matching patterns present in mature tissues may be critical for successful musculoskeletal tissue engineering. Our long-term goal is to develop a bioreactor system in which complex fibrocartilaginous tissues can be matured in vitro. Rather than designing appropriate heterogeneity via engineered scaffolds that are subsequently seeded with cells, our approach is to begin with a uniform population of pluripotent cells and direct their differentiation and matrix production in vitro using loading patterns that mimic in situ conditions. An advantage of this approach is that the bioengineered implants will be conditioned and adapted to those mechanical stresses they will be exposed to after implantation. For this strategy to be successful, we must first characterize the mechanoplasticity (responsiveness to mechanical stimulation) of pluripotent cells as a function of their differentiation stage, and then develop predictive mathematical models. To achieve this, we propose three specific aims. First we will characterize the time-dependent differentiation of human mesenchymal stem cells in 3-dimensional gels under appropriate biologic mediators. Next, we propose to determine at which differentiation stage mesenchymal stem cells respond maximally to either tensile strain or hydrostatic stress and define and validate a mathematical criterion relating matrix loading to cell function. Finally, we will utilize finite element simulations along with the mathematical criterion to define a loading regimen that produces a spatial distribution of matrix deformation (indentation) to pattern cell phenotype and matrix composition. This loading regimen will be applied to gels with mesenchymal stem cells to demonstrate whether spatial heterogeneity of mechanical stimulation can be used to drive spatial heterogeneity in cell phenotype and matrix content in vitro. We anticipate that the results of this research will not only lead to improved tissue engineering methodologies but also provide insight into mechanobiologic processes that guide tissue remodeling and repair.